Novel therapies are needed to'address the vascular endothelial cell (EC) barrier disruption which occurs in the context of inflammatory diseases such as acute lung injury (ALI). We have previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (SIP) and a structurally similar compound, FTY720 (FTY), in models of ALI. Despite its impressive potential, S1P is an endogenous compound that produces a myriad of potentially harmful effects which will limit its usefulness in patients. Therefore we have focused on FTY, which is currently being evaluated in Phase III clinical trials for other indications and may soon be a potential therapeutic option for ALI. Our prior studies demonstrate that FTY potently enhances EC barrier function through a novel and poorly understood mechanistic pathway that differs from SIP. However, FTY has immunosuppressive properties that may limit its therapeutic utility in patients with ALI. As a result, we have generated novel analogues of FTY to mechanistically explore these barrier-regulatory pathways and to identify more optimal therapeutic compounds. Our exciting preliminary data indicate that the FTY720 (S)-phosphonate (fTyS) analogue has superior efficacy in ALI models and maintains SIPRI receptor levels unlike other agonists which induce its ubiquitination and degradation. With this background, we propose the following specific aims. SA #1 will rigorously characterize the differential effects of the promising fTyS analogue in comparison with FTY and SIP on SIPRI expression, activation, ubiquitination, and degradation in vitro. SA #2 will mechanistically characterize in vitro fTyS in comparison with FTY and SIP for their relative effects on multiple aspects of EC barrier function in vitro, including intracellular signaling and junctional complex formation. SA #3 will extend these studies in vivo by defining pharmacologic properties of fTyS in mice and characterizing its effects on SIPRI expression and function in vivo. The relative effectiveness of fTyS, SIP, and FTY to attenuate lung injury will be compared in both short term (LPS) and long term (bleomycin) mouse ALI models to assess the potential use of these agents for prolonged therapy. We expect these studies to facilitate the development of targeted therapies to reduce vascular leak in ALI.